There is current interest in techniques for identifying the presence of cells, e.g., in a sample, such as a cancer biopsy, microbes in body fluids, medical products, foods, and the like. In many situations, it may be desirable to analyze viable (e.g., live) cells and nonviable (e.g., dead) cells. It may also be desirable to distinguish between viable and non-viable cells. Further, it may be desirable to count and/or distinguish such cells simply and quickly.
Traditional cell culture methods for assessing viable and nonviable cells can take hours to days to perform, which can depend upon the organisms that are being tested. Further, in order to separately analyze viable and non-viable cells, complex instrumentation and time-consuming data manipulation may be employed. For example, a variety of systems have been developed that label viable cells with one fluorescent dye and label non-viable cells with both the first dye and a second, different fluorescent dye. To separately analyze the viable and non-viable cells, illumination and detection may use multiple scans using specialized light sources and/or filters to provide and receive selected bands of light. Additionally, the fluorescence received from each dye may be manipulated in software to separately view the viable cells from the non-viable cells.
Viability dyes are known that may be activated within viable cells, for example, by metabolic activity (e.g., esterase cleavage of ester functionalized pro-fluorophores) within the cells. The fluorescent background may be reduced by using a membrane-impermeable quencher, but in such an approach, only the viable cells are detected. Some fluorescent dyes are membrane-impermeable and preferentially label non-viable cells over viable cells. Some systems employ a combination of one or more membrane-permeable dyes, one or more quenchers, and one or more membrane-impermeable dyes. However, increasing amounts of the various dyes and quenchers may undesirably perturb the behavior of viable cells or even kill them, and may also increase the generation of hazardous waste.
The present application appreciates that conducting fluorescent cell-based assays may be a challenging endeavor.